The present invention relates to a method for assaying the activity of creatine kinase (CK) isozyme and to an assay reagent therefor. More particularly, the present invention relates to a method for assaying the activity of creatine kinase MB isozyme (CK-MB) and/or the activity of creatine kinase isozyme localized in mitochondria (mCK) and to an assay reagent therefor.
Human CK includes four proteins whose genes are different. They consist of two kinds of proteins originated from cytoplasms [muscle-type (M-type) and brain-type (B-type) depending on where they are localized] and two kinds of proteins originated from mitochondria, sarcomeric CK (smCK) and ubiquitous CK (umCK). smCK exists in heart muscles and skeletal muscles while umCK is present in small intestines, brains, and stocmachs. The CK isozymes originated from cytoplasms consist of dimers and are classified into 3 types, CK-MM, CK-MB, and CK-BB [Y. Takagi, R. Uzawa, and K. Gomi: Rinsho Kensa (Journal of Medical Technology), Vol. 32, 1309-1315 (1988)]. mCK, which exists in the form of octamer in tissues, is converted with time to dimers in blood. Hereinafter, mCK is meant to include smCK and/or umCK.
The mobilities of the isozymes in electrophoresis are arranged in the order of mCK (octamer), and mCK (dimer)=CK-MM, CK-MB, CK-BB from the cathode. smCK and umCK have equivalent mobilities. Since mCK (dimer) shows the same mobility as that of CK-MM, mCK (dimer) in stored blood is measured as CK-MM by electrophoresis. Besides, there are macro-CKs, which consist of CK and an immunoglobulin connected thereto although they are not classified into isozymes. They can be confirmed from zymograms by mobility, an immune countercurrent method. The properties of the isozymes of CK are shown in Table 1.
In clinical tests, determination of total CK or CK-MB has been widely performed. Particularly, CK-MB is important as a marker for myocardial infarction. Determination methods of CK-MB include an EIA method, an immunological inhibition method, an electrophoretic method, etc.
The EIA method can measure only CK-MB with high specificity whereas it requires a special apparatus and is not quick. The electrophoretic method is cumbersome to operate and requires expertness. It is necessary to obtain the ratio of CK-MB by means of a densitometer before results can be obtained so that this method is not quick either. The immunological inhibition method is advantageous in that quick and easy measurement can be performed by means of an automatic analyzer. However, it has a disadvantage that it lacks specificity.
However, currently, early stage diagnosis of acute myocardiac infarction (hereinafter, sometimes referred to as AMI for short) is desired, so that an immunological inhibition method has been widely used. This method uses an inhibitory antibody to human CK-M subunit to deactivate the M subunit and determines the activity of the remaining B subunit. Use of the method results in measurement of unintended CK-BB and mCKs (dimer and octamer) as well as CK-MB. Of these, CK-BB, which scarcely exists in blood, may be neglected. In addition, there are not so many diseases which involve exudation of it (release of it from the tissue into blood by the destruction of tissue or the like). However, mCK is contained in the sera of healthy persons in amounts substantially the same as CK-MB in terms of activity [Y. Toyoda, et al.: Seibutsu Butsuri Kagaku (Japanese Journal of Electrophoresis), Vol. 42, 175-179 (1998) and T. Hoshino, et al., Seibutsu Butsuri kagaku (Japanese Journal of Electrophoresis), Vol. 42, Supplement No. 2, 21 (1998)]. Furthermore, the exudation of mCK occurs in the case of necrosis of cells as in liver diseases, malignant tumors, etc., which confuses the judgment of the results. Recently, it has been reported that the exudation of mCK occurs in enteritis due to rotavirus, infant apnea, etc. [T. Hoshino, et al.; Rinsho Byori (The Japanese Journal of Clinical Pathology), 46, Plenary Assembly Issue, 57 (1998) and F. Kanemitsu, et al.; Rinsho Bori (The Japanese Journal of Clinical Pathology), 46, Plenary Assembly Issue, 56 (1998)]. In addition, by this measurement method, about half the CK-MB activity is also inhibited and hence the activity is calculated by multiplying the measured value by 2. This doubles the influence of mCK activity. That is, the method of selectively measuring the enzymatic activity of CK isozyme by the conventional immunological inhibition method measures CK-MB activity by using an anti-human CK-M inhibitory antibody and enables convenient and quick measurement. However, this method also measures the activity of mCK simultaneously so that no accurate measurement of CK-MB activity can be expected.
An object of the present invention is to provide a method for measuring the activity of CK-MB which is free of the influence by mCK, accurate, high in specificity, and convenient and which permits automation if desired, by selectively and immunologically eliminating the enzymatic action of mCK. Another object of the present invention is to provide a measurement reagent for use in the measurement method.
As a result of extensive investigation, it has been found that the above objects can be achieved by measuring the activity of CK after treatment of selectively and immunologically eliminating the enzymatic actions of CK-M subunit and mCK, thus completing the present invention.
That is, in one aspect, the present invention relates to a method for measuring the activity of CK-MB isozyme comprising selectively measuring the enzymatic activity of CK isozymes by an immunological inhibition method, wherein after a treatment of selectively and immunologically eliminating the enzymatic actions of CK-M subunit and mCK is performed, the activity of remaining CK is measured.
In another aspect, the present invention relates to the method for measuring the activity of CK-MB isozyme as described above, wherein the treatment of selectively and immunologically eliminating the enzymatic actions of CK-M subunit and mCK is a treatment in which an inhibitory antibody to the CK-M subunit and an inhibitory antibody to the mCK are used.
In still another aspect, the present invention relates to the method for measuring the activity of CK-MB isozyme as described above, wherein the inhibitory antibody to the CK-M subunit and the inhibitory antibody to the mCK are simultaneously acted in one step.
In yet another aspect, the present invention relates to the method for measuring the activity of CK-MB isozyme as described above, wherein the inhibitory antibody to the CK-M subunit and the inhibitory antibody to the mCK are acted in different steps.
In a further aspect, the present invention relates to the method for measuring the activity of CK-MB isozyme, wherein the inhibitory antibody to the CK-M subunit is acted and measurement of the activity of CK is performed and then the inhibitory antibody to the mCK is acted and the measurement is performed again to simultaneously achieve measurement of the activity of mCK.
In a still further aspect, the present invention relates to a method for measuring the activity of mCK comprising measuring the activity of CK, acting an inhibitory antibody to mCK, and again performing the measurement.
In a yet further aspect, the present invention relates to a measurement reagent for measuring CK isozymes, comprising a reagent necessary for a measurement method as described above as a kit or as a single item.